CN102159234A - Treatment of rheumatoid arthritis with mammal beta defensins - Google Patents

Abstract

The present invention relates to treatment of rheumatoid arthritis with mammal beta defensins.

Description

Use the treatment of the rheumatoid arthritis of mammal beta-defensin

Relate to sequence table

The application contains the sequence table that is computer-reader form.This computer-reader form is incorporated herein by carrying stating.

Background of invention

Invention field

The present invention relates to prevent and treat rheumatoid arthritis by using human beta-defensin.

Background

In many other elements, the key component of innate immunity shows quite big selectivitys but collective can kill the antimicrobial peptide (AMP) of broad-spectrum antibacterial, virus and fungus fast for indivedual.The biological importance of AMP is more outstanding because of its widespread distribution at occurring in nature, and it might be produced by all multicellular organisms.In the mankind, advantage AMP is an alexin.Human alpha-defensin is the little peptide of cation, can be divided into α-alexin and beta-alexin based on the topology of its three intramolecularly cysteine disulfide bond.α-alexin can further be subdivided at first from the α-alexin (HNP1-4) of neutrophil cell particle separation and α-alexin (HD5 and HD6) of being expressed in the small intestinal crypts by the Paneth cell.Beta-alexin is mainly produced by the epithelial cell in multiple tissue and the organ, and described tissue and organ comprise skin, trachea, gastrointestinal tract, genitourinary system, kidney, pancreas and mammary gland.(best characterized) member of the tool feature of beta-alexin family is hBD1-3.Yet, use multiple bioinformatics instrument, in the people's gene group note almost 40 codings infer the open reading frame of beta-alexin congener.Some alexins are that composing type produces, and other is inductive by pro-inflammatory cytokine or inoculating microbe product.

Be clear that more and more that except that direct antimicrobial acivity, human alpha-defensin also has the immunomodulating/alternative character of wide region.Described character comprises induces multiple chemotactic factor and cytokine, chemotactic activity and apoptosis activity, induce prostaglandin, histamine and leukotriene to discharge, suppress complement, stimulate dendritic cell maturation by the conduction of tongued bell sample receptor (toll-like receptor) signal, and stimulate pathogen removing by neutrophil cell.In addition, human alpha-defensin also takes place to work in (vasculogenesis) at wound healing, epithelium and fibroblasts proliferation, angiogenesis (angiogenesis) and blood vessel.

The evidence that has increasing human alpha-defensin in many infectivities and inflammatory diseases, to play a significant role.Usually observe the mistake of human alpha-defensin in inflammation and/or the skin infection and express, be likely because induce by microorganism component or endogenous pro-inflammatory cytokine part.In psoriasis (psoriasis), hBD2 and hBD3 are too sufficient, and observe the remarkable rise of hBD2 in the patient's who suffers from acne vulgaris (acne vulgaris) or shallow folliculitis (superficial folliculitis) impaired epithelium.On the other hand, atopy/atopic dermatitis (topic dermatitis) is relevant with the downward modulation of hBD2 and hBD3.

Rheumatoid arthritis is a kind of chronic, general inflammation disease, and it can influence many tissues and organ, but mainly attacks the joint, produces usually to develop into the destruction of articular cartilage and the stiff inflammatory synovitis in joint.Rheumatoid arthritis also can produce diffusivity inflammation and nodositas infringement in lung, pericardium, pleura and sclera, and it is the most common in the subcutaneous tissue under the skin.Though rheumatoid arthritis is agnogenio, autoimmune is played the part of important role in its chronicity and progress.

Detailed Description Of The Invention

Definition

Alexin: term as used herein " alexin " means that those skilled in the art generally acknowledges peptide more than the alexin class belong to antimicrobial peptide.For determining that whether polypeptide is the alexin according to the present invention, can aminoacid sequence and PFAM data base's hidden Markov model preface type (hidden markov model profile) (HMM preface type) be compared by using free available HMMER software kit.

PFAM alexin family comprises for example alexin-1 or " mammal alexin " (accession number PF00323) and alexin _ 2 or alexin _ β or " beta-defensin " (accession number PF00711).

The present invention's alexin belongs to the beta-defensin class.The alexin of beta-defensin class is enjoyed common architectural feature, as the cysteine pattern.

The example of the alexin according to the present invention comprises human beta-defensin 1 (hBD1; Referring to SEQ ID NO:1), human beta-defensin 2 (hBD2; Referring to SEQ ID NO:2), human beta-defensin 3 (hBD3; Referring to SEQ ID NO:3), human beta-defensin 4 (hBD4; Referring to SEQ ID NO:4) and mice beta-defensin 3 (mBD3; Referring to SEQ ID NO:6).

Homogeneity: between two aminoacid sequences or the dependency between two nucleotide sequences describe by parameter " homogeneity ".

For the present invention, by using as EMBOSS software kit (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends in Genetics 16:276-277; Http:// emboss.org), performed Needleman-Wunsch algorithm (Needleman and Wunsch in the Needle program of preferred 3.0.0 version or renewal version, 1970, J.Mol.Biol.48:443-453) determine two homogeneity degree between the aminoacid sequence.The optional parameter that uses extends point penalty (0.5) and EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix as the open point penalty (10) of breach, breach.The output (acquisition of use-nobrief option) of using Needle labelling it " the longest homogeneity " is as percentage ratio homogeneity and following calculating:

(identical residue * 100)/(the breach sum in comparison length-comparison).

For the present invention, by use as the EMBOSS software kit (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, the same; Http:// emboss.org), preferred 3.0.0 version or upgrade Needleman-Wunsch algorithm performed in the Needle program of version (Needleman and Wunsch, 1970, the same) and determine two homogeneity degree between the deoxyribonucleotide sequence.The optional parameter that uses is the open point penalty (10) of breach, breach expansion point penalty (0.5) and EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix.The output (acquisition of use-nobrief option) of using Needle labelling it " the longest homogeneity " is as percentage ratio homogeneity and following calculating:

(identical deoxyribonucleotide * 100)/(the breach sum in comparison length-comparison).

Term as used herein " through separating variant " or " through isolated polypeptide " mean variant or the polypeptide from the source separation.On the one hand, measure as SDS-PAGE, variant or polypeptide are pure at least 1%, and preferably at least 5% is pure, more preferably at least 10% is pure, and more preferably at least 20% is pure, and more preferably at least 40% is pure, more preferably at least 60% is pure, even more preferably at least 80% pure, and most preferably at least 90% pure.

Basically pure polypeptide: term " pure basically polypeptide " represents to contain by weight at the most 10% in this article, preferred at the most 8%, more preferably at the most 6%, more preferably at the most 5%, more preferably at the most 4%, more preferably at the most 3% in addition more preferably at the most 2%, most preferably at the most 1% and even the polypeptide prepared product of 0.5% natural with it or other peptide material that reorganization is relevant at the most most preferably.Therefore, preferred pure basically polypeptide is counted at least 92% pure by the weight of total peptide material existing in the prepared product, preferably at least 94% is pure, more preferably at least 95% is pure, more preferably at least 96% is pure, and more preferably at least 97% is pure, and more preferably at least 98% is pure, even more preferably at least 99% pure, most preferably at least 99.5% is pure and even most preferably 100% pure.Peptide preferably is pure in fact form more than the present invention.This can be for example by by well-known recombination method or prepare polypeptide by classical purification process and realize.

The mammal beta-defensin

The present invention relates to the medical usage of mammal beta-defensin (as human beta-defensin and/or mice beta-defensin) in the treatment rheumatoid arthritis.Treatment TNF-alpha active preferred and in the reduction institute treated tissue is relevant.

In one embodiment, the arbitrary aminoacid sequence among the present invention's mammal beta-defensin and SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and/or the SEQ ID NO:6 has at least 80%, preferred at least 85%, more preferably at least 90% and most preferably at least 95% homogeneity degree.In a preferred embodiment, the arbitrary aminoacid sequence among the present invention's mammal beta-defensin and SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and/or the SEQ ID NO:4 has at least 80%, preferred at least 85%, more preferably at least 90% and most preferably at least 95% homogeneity degree.In a more preferred, the present invention's mammal beta-defensin is made up of the variant (SEQ ID NO:5) and/or the mice beta-defensin 3 (SEQ ID NO:6) of human beta-defensin 1 (SEQ ID NO:1), human beta-defensin 2 (SEQ ID NO:2), human beta-defensin 3 (SEQ ID NO:3), human beta-defensin 4 (SEQ ID NO:4), human beta-defensin 4.One in addition more preferred in, the present invention's mammal beta-defensin is made up of human beta-defensin 1 (SEQ ID NO:1), human beta-defensin 2 (SEQ ID NO:2), human beta-defensin 3 (SEQ ID NO:3) and/or human beta-defensin 4 (SEQ ID NO:4).

In another embodiment, the aminoacid sequence of the present invention's mammal beta-defensin and SEQ ID NO:2 has at least 80%, preferred at least 85%, more preferably at least 90% and most preferably at least 95% homogeneity degree.In a preferred embodiment, the present invention's mammal beta-defensin is made up of human beta-defensin 2 (SEQ ID NO:2).

In yet another embodiment, the present invention's mammal beta-defensin is made up of human beta-defensin and/or mice beta-defensin and functionally equivalent variant thereof.Preferably, the present invention's mammal beta-defensin is made up of human beta-defensin 1, human beta-defensin 2, human beta-defensin 3, human beta-defensin 4 and mice beta-defensin 3 and functionally equivalent variant thereof.More preferably, the present invention's mammal beta-defensin is made up of human beta-defensin 2 and functionally equivalent variant thereof.

The present invention's mammal beta-defensin is also referred to as the chemical compound of preferred embodiment.

Hereinafter, mammal (for example people) beta-defensin it " functionally equivalent variant (functionally equivalent variant) " is for representing modified mammal (for example people) beta-defensin with the roughly the same effect of parent mammal (for example people) beta-defensin to rheumatoid arthritis on the present invention.Preferably, it also represents and the roughly the same effect to the TNF-alpha active of mammal (for example people) beta-defensin.

According to the present invention, the functionally equivalent variant of mammal (for example people) beta-defensin compares with mammal (for example people) beta-defensin aminoacid sequence that can to comprise 1-5 amino acid modified, preferred 1-4 is individual amino acid modified, more preferably 1-3 is amino acid modified, most preferably 1-2 amino acid modified and especially 1 amino acid modified.

Term " modification " means any chemical modification of mammal (for example people) beta-defensin in this article.Modification can be replacement, disappearance and/or the insertion of aminoacid and the displacement of amino acid side chain; Or in aminoacid sequence, use alpha-non-natural amino acid with similar characteristics.In detail, modify and can be amidatioon, as the C-terminal amideization.

Preferably, amino acid modified is more unessential in nature, and it is the folding of not appreciable impact polypeptide and/or the conservative amino acid replacement or the insertion of activity; Single disappearance; Amino-or carboxyl-terminal little extension; The little joint peptide of about at the most 20-25 residue; Or by changing the little extension that net charge or another function help purification, as poly histidine mark, epitope or in conjunction with the territory.

The conservative example that replaces is the replacement in basic amino acid (arginine, lysine and histidine), acidic amino acid (glutamic acid and aspartic acid), polar amino acid (glutamine and agedoite), hydrophobic amino acid (leucine, isoleucine and valine), aromatic amino acid (phenylalanine, tryptophan and tyrosine) and p1 amino acid (glycine, alanine, serine, threonine and methionine) group.Usually not changing than the aminoacid replacement of living is known in this area, and by, for example, Neurath and Hill, 1979 at The Proteins, and Academic Press describes among the New York.The most common generation is exchanged for Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.

Except 20 standard amino acids, non-standard amino acid (as 4-Hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, 2-amino-3-methylpentanoic acid and Alpha-Methyl serine) can replace the amino acid residue of wild type peptide.The non-conserved amino acid of limited quantity, can't help genetic code amino acids coding and alpha-non-natural amino acid can the substituted amino acid residue." alpha-non-natural amino acid " through modifying, and/or has the chemical constitution that is different from primary amino acid at their side chain behind protein synthesis.Alpha-non-natural amino acid can chemically synthesize, and it is preferably commercial available, comprise pipecoliacid (pipecolic acid), Thiazolidine carboxylic acid (thiazolidine carboxylic acid), dehydroproline, 3-and 4-methylproline, with 3,3-dimethyl proline.

Can be according to methods known in the art, for example site-directed mutagenesis or alanine subregion mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085) are identified the essential amino acids in the mammal beta-defensin.In one technology of back, single alanine mutation is incorporated into each residue in the molecule, and the biological activity of the test gained mutating molecule activity of resisting rheumatoid arthritis (that is, to) is to identify the active crucial amino acid residue for described molecule.Equally referring to Hilton etc., 1996, J.Biol.Chem.271:4699-4708.The identity of essential amino acids (identity) also can be by analyzing and being relevant to more than the mammal beta-defensin homogeneity of peptide and inferring.

Can use known mutation, reorganization and/or reorganization (shuffling) method, be relevant screening technique then, and for example those are by Reidhaar-Olson and Sauer, 1988, and Science 241:53-57; Bowie and Sauer, 1989, Proc.Natl.Acad.Sci.USA 86:2152-2156; WO95/17413; Or WO 95/22625 those disclosed method is carried out and is tested single or multiple aminoacid replacement.Other method that can use comprises fallibility PCR, phage display (for example, Lowman etc., 1991, Biochem.30:10832-10837; United States Patent (USP) 5,223, No. 409; WO 92/06204) and directed mutation (Derbyshire etc., 1986, the Gene 46:145 in zone; Ner etc., 1988, DNA 7:127).

More than the present invention the terminal extension of the N-of peptide can be suitably by 1 to 50 aminoacid, preferred 2-20 aminoacid, especially 3-15 aminoacid is formed.In one embodiment, the extension of N-terminal peptide does not contain Arg (R).In another embodiment, terminal kex2 or the kex2-sample cleavage site that comprises as hereinafter further definition that extend of N-.In a preferred embodiment, the N-end extends to the peptide that comprises at least two Glu (E) and/or Asp (D) amino acid residue, as comprises terminal extension of N-of one of following sequence: EAE, EE, DE and DD.

Method and purposes

Finder's beta-defensin 2 rheumatoid arthritis model of in mice, inducing seriousness of parameter that palliates a disease significantly in 41 days collagen protein; Therefore show as the lateral reactivity that is used for the treatment of the medicine of rheumatoid arthritis.

Therefore the present invention provides the method for treatment rheumatoid arthritis, and this treatment comprises mammal beta-defensin from for example pharmaceutical compositions of effective dose to the experimenter of this treatment of needs that use, is preferably human beta-defensin, and more preferably human beta-defensin 2.Also the supplier is mammal beta-defensin (be preferably human beta-defensin, more preferably human beta-defensin 2), and it is used to make medicine; And mammal beta-defensin (be preferably human beta-defensin, more preferably human beta-defensin 2) is used to make the purposes of the medicine for the treatment of rheumatoid arthritis.Treatment comprises the existing disease of treatment or disease and prevention (prophylaxis or prevention) disease or disease.

Mammal beta-defensin treatability is used for compositions, described compositions is used for using via any conventional route through preparation, conventional route (for example comprises enteral (for example through cheek, per os, per nasal, per rectum), parenteral, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular) or local (for example, in epidermis, intranasal or the trachea).In other embodiments, the compositions of the description herein part that can be used as sustained release implants is used.

In other other embodiment, can utilize suitable vehicle that lyophilization thing stability is provided that the compositions of preferred embodiment is formulated as the lyophilization thing, and then rehydration.

The pharmaceutical composition that contains the mammal beta-defensin can be according to conventional methods, for example by mixing, pelletize, coating, dissolving or freeze-drying process manufacturing.

The pharmaceutical composition of preferred embodiment comprises mammal beta-defensin and pharmaceutically acceptable supporting agent and/or diluent.

The mammal beta-defensin preferably in pharmaceutical composition with the amount of effective treatment rheumatoid arthritis, the amount that preferably has the toxicity that can accept the patient is used.For this treatment, the suitably character of the chemical property of the dosage The compounds of this invention that for example depends on certainly to be used and pharmacokinetic data, individual host, mode of administration and the condition of illness of receiving treatment and seriousness and change.Yet, generally speaking, for than large mammals, for example philtrum obtains gratifying result, indication dosage every day is preferably about 0.001mg/kg body weight to about 100mg/kg body weight, and preferably about 0.01mg/kg body weight is to the 50mg/kg body weight, and more preferably from about the 0.05mg/kg body weight is to the 20mg/kg body weight, and most preferably from about the 0.1mg/kg body weight is to about 10mg/kg body weight, for example uses with as many as one day, two, three or four dosage that separates that takes second place.The chemical compound of preferred embodiment can use and so on like mode of administration and similar dosage usually to than large mammals, and for example the people uses.

In certain embodiments, the pharmaceutical composition of preferred embodiment can comprise mammal beta-defensin (as human beta-defensin), its amount depends on that route of administration is for the about 0.5mg of per unit dosage form or still less to about 1500mg or more, preferred about 0.5,0.6,0.7,0.8 or 0.9mg is to about 150,200,250,300,350,400,450,500,600,700,800,900 or 1000mg, and more preferably from about 1,2,3,4,5,6,7,8,9,10,15,20 or 25mg to about 30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100mg.Yet, in some embodiments, can be preferably than above mentioned lower or higher dosage.Those skilled in the art can easily determine suitable concentration and dosage.

Those skilled in the art knows pharmaceutically acceptable supporting agent and/or diluent.For the compositions that is mixed with liquid solution, the supporting agent that can accept and/or diluent comprise saline and sterilized water, and can randomly comprise antioxidant, buffer agent, antibacterial and other common additives.Compositions also can be mixed with pill, capsule, granule, tablet (coat or do not coat), (injectable) solution, solid solution, suspension, dispersion liquid, solid-state dispersion liquid (form that for example is ampoule, bottle, emulsifiable paste, gel, pastel, inhalant powder, foam, tincture, lip pomade, drop, spray or suppository).Formulation can contain salt, buffer agent, diluent, dispersant and surfactant, binding agent, lubricant and/or the other medicines excipient as known in the art of (except that mammal beta-defensin and other optional active component) carrier, filler, disintegrating agent, flowing regulator, sugar and sweeting agent, aromatic, antiseptic, stabilizing agent, wetting agent, emulsifying agent, solubilizing agent, adjusting osmotic pressure.Those skilled in the art can be further in a suitable manner and according to generally acknowledged maneuver (as Remington ' s Pharmaceutical Sciences, Gennaro compiles, Mack Publishing company, Easton, the maneuver of description among the PA 1990) preparation mammal beta-defensin.

The mammal beta-defensin can use separately or use with the combination treatment of a kind of, two or more other medicines chemical compounds or drug substance and/or one or more pharmaceutically acceptable excipient.

External synthetic

The mammal beta-defensin can use as known in the art conventional method by external synthetic preparation.Multiple commercial synthesizer is available, for example the automatic synthesizer of Applied Biosystems company, Beckman etc.By using synthesizer, available alpha-non-natural amino acid, especially D-isomeric compound (or D-form) (for example D-alanine and D-isoleucine), diastereoisomer, side chain etc. with different length or functional group replace the aminoacid of natural existence.Particular sequence and preparation method will be determined by suitability, economy, required purity etc.

Can provide more than comprising the functional group of being convenient to binding and to plant peptide or the chemistry of protein is connected, described functional group is for amide or be substituted amine to form (for example reducing aminated) speech be amido, for thioether or disulphide formation is mercapto, is carboxyl etc. for amide forms.

If desired, can in peptide, introduce the group that a plurality of permissions are connected with other molecule or surface between synthesis stage or during expressing.Therefore, can use cysteine to make thioether, histidine is used to be connected in the metal ion complex, and carboxyl is used to form amide or ester, and amido is used to form amide and analog thereof.

Also can separate and purification mammal beta-defensin according to the conventional method that is re-combined into.Lysate can be prepared by expressive host, and lysate can use HPLC, exclusion chromatography, gel electrophoresis, affinity chromatograph or other purification technique purification.

The present invention is further described by the following example, and described embodiment should not be construed as restriction the present invention's scope.

Embodiment

Embodiment 1

Human beta-defensin 2 in the rheumatoid arthritis model that the assessment collagen protein is induced

During the immunomodulatory effect of test hBD2, observe hBD2 unexpectedly and have huge antiinflammatory potentiality.

Herein, we have showed that hBD2 has significant effect in mice in the rheumatoid arthritis model that collagen protein is induced in the treatment rheumatoid arthritis.

Human beta-defensin 2 (hBD2)

Reorganization produces hBD2.The synthetic DNA fragment (DNA 2.0) of hBD2 of will encoding is cloned in pET-32 (+) expression vector (Novagen).The plasmid-encoded translation fusogenic peptide of gained, this translation fusogenic peptide contain the terminal thioredoxin part of N-, succeeded by his-labelling, enterokinase cracking site and be the hBD2 peptide at last.Expression plasmid is transformed among escherichia coli (E.coli) the bacterial strain BL21.

To grow to OD600 be about 8 and induced 3 hours with 0.5mM IPTG with 100 times of the overnight culture of this bacterial strain dilutions and at 37 ℃ in the TB-glycerol that contains 100 mcg/ml ampicillin, after this passes through centrifugal collecting cell.Use standard scheme to go up the trx-hBD2 fusogenic peptide of purification his labelling on Ni-NTA pearl (QIAGEN).Subsequently with his-labelling purification fusogenic peptide dialyzed overnight in enterokinase buffer agent (50mM tris-HCl (pH 7.5), 1mM CaCl ₂) in, and with the enterokinase cracking to discharge ripe hBD2.Use Source 15S substrate (Amersham Biosciences) to be further purified the hBD2 peptide by cation-exchange chromatography.Use the correction molecular weight of MALDI-TOF mass spectrum checking hBD2.

Use same approach to produce mBD3 (referring to embodiment 5).

The suitably folding and disulphide bridges topology of the trypsinization checking hBD2 molecule of use subsequently and LC-MS and the coupling of NMR spectroscopy.

Remove endotoxin by preparation type RP-HPLC at low pH value, and measure (Endosafe KTA2) by LAL and measure endotoxin content and find that this level is lower than the detectability of mensuration (0.05EU/mg).For the level of the detectability of determining to be lower than endotoxin measurement can not stimulate PBMC, do with the extremely effective titration curve of lipopolysaccharide (escherichia coli, O111:B4, Sigma L4391) stimulation.Extremely low-level this LPS (0.06ng/ml) can stimulate PBMC to produce the cytokine that can detect.

Below the purpose of research is to determine the anti-inflammatory activity of human beta-defensin 2 in rheumatoid arthritis.

Test macro

Species/strain: mice/DBA/1

Source: Harlan, UK

Sex: male

Animal number: n=50

Age: young become Mus, studying when initial is 6-8 age in week.

Body weight: the weight differential of zoologizeing when collagen protein is induced be no more than average weight it ± 20%.

Animal health condition: the health status of checking the animal of being used in this research during arrival.Only make healthy animal adaptation laboratory condition and be used for research.

Adapt to: at least 7 days.

Settle: between the laundering period and after the administration, be placed in animal in the limited rodent facility of inlet and grouping raise in be equipped with hard-caked sediment and fill wood shavings as the polypropylene cage of laying material (among 45cm * 25cm * 13cm), every group of maximum 10 mices.Change cage weekly once.

Food and water: provide commercial rodent diet and the animal that freely to obtain can freely obtain the drinking water that is supplied to each cage via polyethylene bottle with rustless steel suction pipe (sipper tube) to animal.At least per 3 weeks are changed water bottle.Change water weekly 3 times.

Environment: the setting environmental condition of control automatically maintains 20-24 ℃, and relative humidity (RH) is 30-70%, 12/12 hour illumination/dark cycle, and per hour take a breath 10-30 time in the research room.By manual measurement and Control Computer monitor temperature every day and RH.By Control Computer monitoring illumination circulation.

Identify: give the unique animal of animal and identify the ear numbering.This numbering also is presented on the visible cage card of each cage front end.The cage card also contains the research numbering.

Randomness: with the animal random assortment to experimental group.

Stop: when research finishes, by sucking O ₂/ CO ₂Make surviving animals euthanasia, then blood-letting.

Reason: select mice, because it represents the selected species of this experimental animal model.The DBA/1 strain height of mice is easily suffered from the arthritis (CIA) that collagen protein is induced.

Material

Human beta-defensin 2 (hBD2); Referring to more than

Dexamethasone (Sigma, catalog number (Cat.No.) D1756)

II type bovine collagen albumen (MD Biosciences, catalog number (Cat.No.) 804001314)

Complete Freund's adjuvant (CFA) (MD Biosciences, catalog number (Cat.No.) 501009703)

PBS (PAA, catalog number (Cat.No.) H15-002)

The formation of test group

Table 1: test group and processing

The group scale

Group #

Test compounds

Approach

Dosage

Volume

Regime

n＝10

A

The mediator matched group

IV

0mg/kg

5mL/kg

Once a day

n＝10

B

Dexamethasone

IP

1mg/kg

5mL/kg

Once a day

n＝10

C

hBD2

IV

10mg/kg

5mL/kg

Once a day

n＝10

D

hBD2

IV

1mg/kg

5mL/kg

Once a day

n＝10

E

hBD2

IV

0.1mg/kg

5mL/kg

Once a day

IV: intravenous

IP: intraperitoneal

Test program

Arthritis is induced

Study the 0th day (research beginning), under slight isoflurane anesthesia, use plastic injector to make all animals in afterbody intradermal injection 0.1ml II collagen type/CFA emulsion (every mice 200 microgram collagen protein).Injection position is positioned at roughly back range from the about 1cm of tail root from locating.The 21st day, provide collagen protein to excite (200 μ g/ mice) to animal by peritoneal injection collagen protein and PBS.

Handle

Study the 14th day and begin handle and whole during continue once a day.Study the 42nd day, stop (terminate) all survival mice.

Route of administration:

(i) hBD2: intravenous

(ii) dexamethasone: intraperitoneal

(iii) mediator contrast: intravenous

Dosage and volume dose (also referring to table 1):

(i) hBD2:10,1 or 0.1mg/kg is with 5mL/kg

(ii) dexamethasone: 1mg/kg is with 5mL/kg

(iii) mediator contrast: 0mg/kg is with 5mL/kg

Pain relieving: do not use analgesic during the research.

Observe and inspection

Arthritis reaction

Study the 0th day, the 14th day, the 21st day and after this stop until research for 5 times weekly, check the symptom that causes arthritis antigen-reactive (arthritogenic response) among the Zhou Guanjie outside the mice.According to the arthritis reaction of reporting each pawl by the 0-4 scale of the incremental order of seriousness, as follows:

Clinical symptom

The 0th day, the 14th day, the 21st day and after this carry out careful clinical examination weekly for 5 times and record in addition.Observed result comprises the generation of variation, secretion and drainage (for example diarrhoea) of skin, fur, eyes, mucosa and autonomic activities (for example shed tears, sialorrhea, piloerection, pupil size, unusual breathing pattern).Also write down gait, posture and to the variation and the strange behavior of the reaction of processing, tremble, twitch, sleep and the existence of stupor.

Before the 14th day, monitor any unusual behavior of mice every day.

Body weight

Study the 0th day, the 14th day, the 21st day and after this stop until research for 5 times weekly, before arthritis is induced, measure the whose body weight of animal at once.

The measurement of experiment arthritis

Study the 0th day, reached after this in the 14th day, the 21st day and pass through to use dials slide calliper rule (dial caliper) (Kroeplin weekly for 5 times, Munich, Germany) with mm be the unit two rear solid end thickness of measuring each animal (left side and right side, just below the foot pad with calcaneus above) relative variation.

Research stops

Study the 42nd day, stop all mices.

Sample collection

When research stops, sucking O ₂/ CO ₂After, all residues are zoologizeed and are obtained the terminal blood sample certainly.Prepare serum and be stored in-20 ℃ by each sample.In addition, collect left side fore paw and rear solid end and be stored in the formalin, and collect right side fore paw and rear solid end and quick freezing and analyze to be used for possible joint RNA.

The humanity terminal point

The people is genuine to be made and can find to be in the animal of moribund condition and show severe pain and stand the animal euthanasia of the painful symptom of severe.In addition, the genuine display body anharmonic ratio original weight measured value that makes of people reduces to surpass 20% animal euthanasia.For human reason, also select total arthritis score and be 12 or surpass 12 mice.By sucking O ₂/ CO ₂Make all animal euthanasias, then blood-letting.Zoologize from all and to obtain pawl sample and terminal blood sample.

Statistical analysis

Assessment is mainly based on the meansigma methods and the pawl thickness measurement of arthritis score.In the time of suitably, use suitable statistical method analytical data to determine the significance of Treatment Effects.Use ANOVA, then use the significant difference of telling between basic postmortem analysis (Tukey post-hoc analysis) (Winstat 2005.1 of Excel) the evaluation processed group.

According to the Ministry of Internal Affairs (Home Office) regulation, select total clinical score because of arthritis seriousness and be equal to or greater than 12 mice.For because of removing high scoring mice people, during the clinical score when described mice is stopped continues on for remaining and researchs and analyses for twisting data.

Animal is looked after and uses and handle

Carry out this research according to British Home Office about the regulation of using animal in the scientific procedure.

The result

Show the average clinical arthritis score in the male DBA/1 arthritis mice that collagen protein is induced, determined during 2:42 days observation periods.* p＜0.05 significantly is different from the mediator group.

Conclusion

The arthritis reaction of record research all groups since the 14th day.The peak value that mediator is handled average total arthritis score (table 2) of mice was 8.5 ± 0.72 on the 41st day in research.Peak value through average total arthritis score of the mice (C group) of 10mg/kg hBD2 processing was 5.0 ± 1.04 on the 40th day in research.Finished until research from the 23rd day, the average arthritis score of this group is compared low with mediator processing mice, yet only remarkable in the time of the 41st day in research.

Through the peak value of average total arthritis score of the mice (D group) of 1mg/kg hBD2 processing research the 41st day be 5.2 ± 1.11 and finished to compare until research from the 21st day with the mediator processed group lower always, yet only remarkable in the time of the 40th day.Compare with the mediator processed group, handle mice (E group) with 0.1mg/kg hBD2 and significantly do not reduce average total arthritis score.The peak value of the average score of this group was 9.0 ± 0.77 on the 40th day in research.Finish until research from studying the 26th day, the mice in the dexamethasone processed group (B group) is compared with the mediator processed group and shows significantly lower arthritis score.

For guarantee mice that research is selected because of arthritis seriousness in early days remove not can the people for twisting data, during the arthritis score of these mices continued on for analyzing until the research termination.

Embodiment 2

The anti-inflammatory activity of human beta-defensin 2 (hBD2)

In human PBMC's culture, observing with the hBD2 treatment stimulates the cytokine spectrum of culture to produce significant impact to LPS, LTA or Peptidoglycan.Before observe hBD2 and can induce pro-inflammatory cytokine and chemotactic factor IL-6, IL-1 β, RANTES, IP-10 and IL-8 (Niyonsaba etc., 2007; Boniotto M. etc., 2006).

Herein, we show that hBD2 has the downward modulation potentiality to two kinds of pro-inflammatory cytokine TNF and 1L-1 β; And hBD2 also induces IL-10 after ester polysaccharide (LPS), lipoteichoic acid (LTA) or Peptidoglycan (PGN) are induced inflammatory stimulus.IL-10 be potential anti-inflammatory cytokines and therefore the effect of gained hBD2 be anti-inflammatory effect.Human PBMC, mononuclear cell cell line and class dendritic cell systems (dendritoid cell line) have observed this result.

HBD system as in preparation described in the embodiment 1.

Separate and stimulation PBMC

Extract peripheral blood from healthy volunteer's (through approval of the relevant Ethics Committee of Denmark).With RPMI 1/1 (v/v) dilution heparinized blood, and in 2 hours that extract, make it to carry out the Ficoll density centrifugation.Layer is collected blood plasma and is remained on ice and is used for culture medium (from the body culture medium) until it with 2% on indivedual donors.To be resuspended in the body culture medium and be inoculated in 96 well culture plates through separating PBMC, 255,000 cells in every hole, cumulative volume is 200 microlitres.With 100,10 or 1 μ g/ml hBD2 stimulate separately or with 0.6ng/mL or 20ng/mL LPS (escherichia coli, O111:B4, Sigma L4391), 1.25 μ g/ml lipoteichoic acids (LTA) are (from bacillus subtilis (B.subtilis), Sigma L3265) or 40 μ g/ml Peptidoglycans (PGN) (from staphylococcus aureus (S.aureus), Sigma 77140) stimulate PBMC together from identical donor.In initial experiment, be used for the concentration of stimulation,, use two kinds of variable concentrations to be in the cytokine levels that to regulate to guarantee it for LPS at 3 kinds of different donor optimizations.In some experiments, handle the contrast of PBMC separately and with LPS or LTA as the downward modulation inflammatory cytokine with dexamethasone and indomethacin.Collect supernatant at 37 ℃ of incubations after 24 hours, and be stored in-80 ℃ until measuring cytokine.All the experiment in by Alamar Blue (Biosource, DALL1100) measure viability, and also measure viability by MTS (Promega) in some cases, and in some experiments, also by judging with the Nucleocounter counting cells according to the description of manufacturer.

Cultivate and stimulation MUTZ-3

The cell line MUTZ-3 that people's myeloid leukemia is derived (DSMZ, Braunschweig, Germany) maintain be supplemented with 20%[volume/volume (v/v)] hyclone (Sigma F6178) and 40ng/mlrhGM-CSF (R﹠amp; D Systems 215-GM-050) among the a-MEM (Sigma M4526).Mononuclear cell cell line and described mononuclear cell that described CFU-GM is arranged in following explanation stimulate separately with 100,10 or 1 μ g/ml hBD2 or stimulate with LPS or LTA.

Dendritic cell differentiation

For producing class dendritic cell system, making people's bone marrow leukemia cells is MUTZ-3 (1 * 10 ⁵Individual cells/ml) in the presence of rhGM-CSF (150ng/ml) and rhlL-4 (50ng/ml), is divided into immature DC and lasts 7 days.Changed culture medium in every 2-3 days.Further stimulate the cell line of differentiation to explore the effect of hBD2 with LPS or LTA to dendritic cell in the hBD2 existence or not.

Cytokine measurements

On the FACSarray flow cytometer, measure the generation of cytokine in the supernatant by flow cytometry according to description (BD) the personnel selection inflammatory cells counting pearl array (CBA) of manufacturer.Measure following cytokine: IL-8, IL-1 β, IL-10, TNF, IL-12 p70, IL-6.In some experiments, according to the description of manufacturer by R﹠amp; The ELISA cover group of D systems (IL-10, TNF-α, IL-1 β) is measured cytokine.

Data analysis

All experiments carry out at least twice, show representative result.The data that presented can mean+/-standard deviation (SD) expression.Being treatment (hBD2, dexamethasone etc.) and being stimulated double factor (2-way) ANOVA of (LPS, LTA, Peptidoglycan etc.) by variable, is that statistical significance is determined in check (post-test) behind the Bonferroni then, as reporting in the table mark.P＜0.05 is considered as significant difference.

The result

Test hBD2 to through or without the human PBMC's of LPS and LTA treatment effect (table 1,2 and 3).The treatment of hBD2 makes the TNF in the stimulation culture of all three test concentrations significantly reduce (table 1), and this downward modulation is to the LPS of 0.6ng/ml and to mention LTA be dose-dependent.For IL-1 β, mainly under maximum dose level, observe downward modulation (table 2).Noticeable is that the IL-10 dose dependent significantly raises (table 3).The extremely strong antiinflammatory potentiality of inducing demonstration hBD2 of accent and anti-inflammatory cytokines under the pro-inflammatory cytokine.Measure viability to get rid of the anti-inflammatory effect of hBD2 by two kinds of different mensuration owing to cytotoxic effect.In table 4 and table 5, visible hBD2 not pair cell produces cytotoxic effect, observed to effect be stimulating effect owing to the stimulation of LPS that causes cell proliferation or LTA.Therefore, hBD2 does not have cytotoxic effect to described cell.

In table 8,9 and 10, by ELISA rather than by the cytokine of flow cytometry, observe identical result herein, but the sensitivity of mensuration is lower and detectability is much higher, and therefore effect is not remarkable with clear liquid on another donor of cytometer beads array analysis.

For testing another tongued bell sample receptors ligand, research hBD2 is to the effect (table 9 and 10) of the PBMC of Peptidoglycan stimulation.Observe identical result: downward modulation of TNF dose dependent and IL-10 dose dependent are induced.

As the positive control of TNF downward modulation, test two kinds of anti-inflammatory compounds, dexamethasone and indomethacins in mensuration.Select concentration so that chemical compound is nontoxic and the concentration for realizing owing to the dissolubility in the culture medium.After LTA stimulated, only indomethacin suppressed TNF (table 11), and dexamethasone is effectively reduced the TNF generation, and IL-1 β also observes identical result (table 13).Indomethacin is COX-1 and COX-2 mortifier and for being used for the treatment of slightly to the nonsteroid anti-inflammatory drugs (NSAID) of moderate pain and helping the symptom of ameliorate osteoarthritis, and dexamethasone has pro-inflammatory cytokine under very low dose and extremely effectively reduces effect (Rowland etc. for being mainly used in the treatment synthetic glucocorticoid of inflammatory disease and its, 1998), for TNF-α and IL-1 β, we also observe identical result.Two kinds of anti-inflammatory compounds of hBD2 and this are the same effective or better than it.

In table 14 and 15, shown the effect of hBD2 to transferring TNF and dendritic cell under in the mononuclear cell cell line, PBMC also observed identical result.Dendritic cell through hBD2 and LPS or hBD2 and LTA stimulation are also induced IL-10 (result does not show).

Transfer the effect that test hBD2 stimulates the PBMC due to the synthetic ligands (Pam3CSK4 (TLR2-TLR1 part), InvivoGen tlrt-pms) under getting rid of hBD2 and combining of LPS or LTA causing TNF and IL-1 β.Behind this ligand stimulation, hBD2 also can reduce TNF, shows among LPS or the LTA and can not cause viewed effect (result does not show).In addition, the cytokine mixture (cocktail) that contains TNF-α and IL-α together with hBD2 to the stimulation of dendritic cell with compare 1L-1 β and IL-8 and IL-6 with the stimulation of cytokine mixture separately and have the downward modulation effect.Obviously, do not analyze the effect to TNF (result does not show) owing to the stimulation of TNF-α.

Table 3: after LPS or LTA processing, produce TNF from human peripheral blood mononuclear cell (PBMC) in the presence of the hBD2 existence reaches not, all specimen needle are tested identical donor, come from the representativeness experiment of 5 donors.Measure TNF by cytometer beads array (CBA) on FACSarray, * * * refers to analyze (N=of each data set about 200) by double factor ANOVA, compares p＜0.001 with contrast (runic) separately.

Table 4: after LPS or LTA processing, produce IL-1 β from human peripheral blood mononuclear cell (PBMC) in the presence of the hBD2 existence reaches not, all specimen needle are tested identical donor, come from the representativeness experiment of 5 donors.Measure IL-1 β by cytometer beads array (CBA) on FACSarray, * * * refers to analyze (N=of each data set about 200), p＜0.001 by double factor ANOVA.

Table 5: after LPS or LTA processing, produce IL-10 from human peripheral blood mononuclear cell (PBMC) in the presence of the hBD2 existence reaches not, all specimen needle are tested identical donor, come from the representativeness experiment of 5 donors.Measure IL-10 by cytometer beads array (CBA) on FACSarray, * * * refers to analyze (N=of each data set about 200) by double factor ANOVA, p＜0.001, and * * refers to p＜0.01, * refers to p＜0.5.

Table 6: by the viability that stimulated back PBMC in 24 hours of MTS calibrating measurement.By double factor ANOVA, then by Bang Fulangni check afterwards, the value difference that has different subscript letter in each row is different significantly.

Table 7:, one of come from 5 experiments of 5 kinds of different donors a representative experiment by the PBMC viability of my Acanthaceous indigo (Alamar Blue) measurement.By double factor ANOVA, then by Bang Fulangni check afterwards, it is different significantly to have the value difference that has different subscript numerals in the value of different subscript letters and each row in each row.

Table 8: the TNF-α of PBMC secretion after hBD2, LTA, LPS or its combination of stimulation.Measure TNF-α by ELISA, nd: do not detect, the detection of mensurations is limited to 0.01ng/ml, and * refers to compare with contrast separately, p＜0.05, and * * refers to compare p＜0.01 with contrasting separately.

Table 9: after hBD2, LTA, LPS or its combination of stimulation, the IL-10 of PBMC secretion is measured TNF-α by ELISA, and nd: do not detect, the detection of mensuration is limited to 0.03ng/mL.

Table 10: after hBD2, LTA, LPS or its combination of stimulation, the IL-1 β of PBMC secretion is measured TNF-α by ELISA, and nd: do not detect, the detection of mensurations is limited to 0.016ng/mL, and * * refers to compare p＜0.01 with contrasting separately.

Table 11: in the hBD2 existence and not, after PGN handles, produce TNF from human peripheral blood mononuclear cell (PBMC), all specimen needle are tested identical donor.Measure TNF by cytometer beads array (CBA) on FACSarray, * * * refers to analyze (N=of each data set about 200) by double factor ANOVA, compares p＜0.001 with contrast separately.

Table 12: in the hBD2 existence and not, after PGN handles, produce IL-10 from human peripheral blood mononuclear cell (PBMC), all specimen needle are tested identical donor.Measure TNF by cytometer beads array (CBA) on FACSarray, * * * refers to analyze (N=of each data set about 200) by double factor ANOVA, compares p＜0.001 with contrast separately.

Table 13:, after LPS or LTA processing, produce TNF from human peripheral blood mononuclear cell (PBMC) in two kinds of different contrasts (dexamethasone and indomethacin) existence of hBD2 or inhibition TNF and not; All specimen needle are tested identical donor.Measure TNF by cytometer beads array (CBA) on FACSarray, analyze (N=of each data set about 200) by double factor ANOVA, compare with contrasting (runic) separately, the value that underlines significantly reduces.

Table 14:, after LPS or LTA processing, produce IL-10 from human peripheral blood mononuclear cell (PBMC) in two kinds of different contrasts (dexamethasone and indomethacin) existence of hBD2 or anti-inflammatory effect and not; All specimen needle are tested identical donor.Measure IL-10 by cytometer beads array (CBA) on FACSarray, analyze (N=of each data set about 200) by double factor ANOVA, compare with contrasting (runic) separately, the value that underlines significantly increases.

Table 15:, after LPS or LTA processing, produce IL-1 β from human peripheral blood mononuclear cell (PBMC) in two kinds of different contrasts (dexamethasone and indomethacin) existence of hBD2 or anti-inflammatory effect and not; All specimen needle are tested identical donor.Measure IL-1 β by cytometer beads array (CBA) on FACSarray, analyze (N=of each data set about 200) by double factor ANOVA, compare with contrasting (runic) separately, the value that underlines significantly reduces.

Table 16:, after LPS or LTA processing, on person monocytic cell's cell line (MUTZ-3), produce TNF in the clear liquid in the hBD2 existence and not.Measure TNF by cytometer beads array (CBA) on FACSarray, * refers to analyze (N=of each data set about 200) by double factor ANOVA, compare with contrast separately, and p＜0.05, * * refers to compare p＜0.01 with contrast separately.

Table 17:, on the immature dendritic cell of LPS or LTA (producing mature dendritic cell (DC)) stimulation, produce TNF in the clear liquid in the hBD2 existence and not.Measure TNF by cytometer beads array (CBA) on FACSarray, * refers to analyze (N=of each data set about 200) by double factor ANOVA, compare with contrast separately, and the p that significantly reduces＜0.05, * * * refers to compare the p that significantly reduces＜0.01 with contrast separately.

Embodiment 3

The anti-inflammatory activity of hBD1, hBD2, hBD3 and hBD4 variant

Basically carry out embodiment 3 as described in example 2 above.Chemical compound rhBD2 as shown in following table is reorganization hBD2, and it is identical with the hBD2 that used among the embodiment 2.

Chemical compound hBD1, hBD2 as shown in following table, hBD3 and hBD4 variant system utilize the chemosynthesis preparation and obtain from Peptide institute Inc.

The aminoacid sequence of reorganization hBD2 (rhBD2) is with identical by the aminoacid sequence of the hBD2 of chemosynthesis preparation.

HBD4 variant shown in the following table is made up of the aminoacid 3-39 of hBD4, and aminoacid sequence is shown in SEQ ID NO:5.

In each table, all specimen needle are to testing identical donor.SD means standard deviation.

The result

Table 18: in monoclonal antibody of the sharp former times existence of human beta-defensin, dexamethasone or English and not, after the LPS treatment, blood mononuclear cell (PBMC) produces TNF slightly from the people end.Measure TNF by cytometer beads array (CBA) on FACSarray, * refers to be analyzed and checked with untreated cell by Bang Fulangni afterwards by double factor ANOVA compare, p＜0.05, and * * refers to p＜0.01, * * * refers to p＜0.001.

Table 19:, after the LPS treatment, produce IL-10 from human peripheral blood mononuclear cell (PBMC) in monoclonal antibody of the sharp former times existence of human beta-defensin, dexamethasone or English and not.Measure IL-10 by cytometer beads array (CBA) on FACSarray, * refers to be analyzed and checked with untreated cell by Bonferroni afterwards by double factor ANOVA compare, p＜0.05, and * * refers to p＜0.01, * * * refers to p＜0.001.

Table 20:, after the LPS treatment, produce IL-1 β from human peripheral blood mononuclear cell (PBMC) in monoclonal antibody of the sharp former times existence of human beta-defensin, dexamethasone or English and not.Measure IL-1 β by cytometer beads array (CBA) on FACSarray, * * * refers to be analyzed and checked with untreated cell by Bonferroni afterwards by double factor ANOVA compare p＜0.001.

Test hBD1, hBD2, hBD3 and hBD4 variant to through or without the human PBMC's of LPS treatment effect (table 18,19 and 20).For comparing, in each situation, comprise rhBD2.

For all alexins, the TNF downward modulation.Reducing of IL-1 β secretion can be suitable with TNF, but it is obvious to be not so good as TNF.For hBD2 and hBD4 variant, the secretion dose dependent ground of IL-10 significantly strengthens.

Also test the hBD3 of 10 μ g/ml and 40 μ g/ml, and also test the hBD4 variant of 40 μ g/ml; Yet, because two kinds of molecules pair cell under described concentration is poisonous, so can not distinguish poisonous effect and anti-inflammatory effect.

As the positive control of TNF downward modulation, comprise two kinds of anti-inflammatory compounds in this case, dexamethasone and English monoclonal antibody of sharp former times.

Conclusion

The human beta-defensin of all tests all shows the antiinflammatory potentiality.

Embodiment 4

The minimizing of deutero-dendritic cell of person monocytic cell and human PBMC's IL-23

Basically as described in human PBMC's embodiment 2, carrying out embodiment 4; Yet reading is IL-23, rather than TNF, IL-1 β and IL-10.In addition, also study the effect of rhBD2 to the deutero-dendritic cell of person monocytic cell.

The generation of the dendritic cell of monocyte derived (DC)

The rules of revising according to initial descriptions such as Romani prepare DC.In brief, by buffy coat (buffy coat) the purification peripheral blood lymphocytes (PBMC) of Ficoll-pague (GE-healthcare) gradient centrifugation from healthy donors.(Dynal, Invitrogen) the positive CD14+ cell of selecting is from the PBMC separating monocytic cell to pass through magnetic bead according to the description of manufacturer.In 6 orifice plates at RPMI/2% people AB serum recombined human reorganization granulocyte-macrophage colony stimutaing factor (GM-CSF, 20ng/ml) and IL-4 (20ng/ml) the CD14+ mononuclear cell was cultivated 6 days the supply medium/cells factor after 2 days and 5 days in (PeproTech).Cultivate after 6 days, cultivate immature DC again in 96 orifice plates, concentration is 1 * 10 ⁶Individual cells/ml, and disregard or handled again 24 hours with mixture and/or hBD2.Test hBD2 in quadruplicate with four kinds of concentration.The inhibition hDC maturation that use contains the scorching mixture analysis of inspiring of LPS (100ng/ml) and IFN-γ (20ng/ml) hBD2 becomes the ability that inspires scorching phenotype.Before mixture 20 hours, add dexamethasone as positive control with chemical compound of the clinical anti-inflammatory activity of verifying.Before adding mixture 4 hours, with the hBD2 incubation.

Cytokine ELISA

Collecting cell culture supernatants and be stored in-80 ℃.Use commercial available antibody and reference material to measure the amount of IL-23 according to the rules (eBioscience) of manufacturer by the standard sandwich ELISA.

The MTT calibrating

Whether use has cell seriously to be subjected to mediator (vehicle), mixture or hBD2 to handle influence based on the cell growth measurement cover group of MTT as measuring with assessment of cell survival after 48 hours, and carries out according to the rules (Sigma) of manufacturer.

Statistical analysis

All experiments carry out at least twice, show representative result.The data that presented can mean+/-standard deviation (SEM) expression.For treatment (hBD2, dexamethasone etc.) and stimulate the double factor ANOVA of (LPS, LTA, Peptidoglycan etc.), is that Bonferroni checks definite statistical significance afterwards by variable then, such as in the table mark report.P＜0.05 is considered as significant difference.

The result

Table 21: with culture medium (stimulate) or LPS and IFN-γ stimulates and through the people CD14 of culture medium (being untreated), hBD2 or dexamethasone processing ⁺IL-23 (pg/ml) in the dendritic cell supernatant of monocyte derived, meansigma methods (SEM), N=4 one of comes from three a representative donor.* refer to analyze and check with untreated cell afterwards compare by Bonferroni by double factor ANOVA, p＜0.05, * * refers to p＜0.01, * * * refers to p＜0.001.Nd: do not detect (being lower than detectability).

Table 22: through culture medium (contrast), 0.6ng/ml LPS, 20ng/ml LPS or 5 μ g/ml LTA stimulation and the IL-23 (pg/ml) in human PBMC's supernatant of the sharp former times monoclonal antibody processing of hBD2, dexamethasone or English, meansigma methods (SEM).* refer to check with untreated cell afterwards and compare by the one-way ANOVA analysis and by Du Shi multiple comparisons (Dunnett ' s Multiple Comparison), p＜0.05, * * refers to p＜0.01, * * * refers to p＜0.001.

As shown in Table 21, the hBD2 dose dependent significantly suppresses IL-23 from people CD14 ⁺The secretion of the dendritic cell of monocyte derived.

For the human PBMC, IL-23 secretes also significantly be inhibited (table 22).To described cell, there is inverse (inverse) dose dependent, when test during, find that it is that bell (bell-shaped) dose-response suppresses curve (data not shown) than the hBD2 of low dosage.

This shows that hBD2 can have depression effect by suppressing the IL-23 secretion in chronic autoimmune condition of illness, because IL-23 is the pith of inflammatory response.The survival and the propagation of Th17 cell depend on IL-23, and have shown that the Th17 cell is pathogenicity in several autoimmune diseases (as Crohn disease, ulcerative colitis, psoriasis and multiple sclerosis).

Embodiment 5

Mice beta-defensin 3 (mBD3) makes TNF reduce from the secretion of PBMC

Basically as described in human PBMC's embodiment 2, carrying out embodiment 5.Use with embodiment 1 in be used for the generation of hBD2 identical rules prepare mice beta-defensin 3 (mBD3).The aminoacid sequence of mBD3 is shown among the SEQ ID NO:6.Preparation mice PBMC as described below.

Separate and stimulation mice peripheral blood lymphocytes (PBMC)

In the blood of ten NMRI mices, separate the mice peripheral blood lymphocytes.In brief, with RPMI1/1 (v/v) dilution heparinized blood, and make it to carry out the Ficoll density centrifugation in back 2 hours in extraction.Collect blood plasma from the upper strata and give up.To be resuspended in the culture medium (RPMI 1640 (Gibco, 42401) with 1% penicillin and streptomycin and 1%L-glutamine) through separating PBMC, and be inoculated in 96 well culture plates, 115,500 cells in every hole amount to 200 μ l.Stimulate separately or stimulate PBMC together with 100,10 or 1 μ g/ml hBD2 or mBD3 (mice beta-defensin 3) from identical donor with 20ng/ml LPS (escherichia coli, 0111:B4, Sigma L4391).Add the 3.5ng/ml dexamethasone to warp or in without the culture of LPS stimulation.Collect supernatant at 37 ℃ of incubations after 24 hours, and be stored in-80 ℃ until measuring cytokine.

On the FACSarray flow cytometer, measure the generation of cytokine in the supernatant by flow cytometry with mice inflammatory cells counting pearl array (CBA) according to the description (BD) of manufacturer.

After collecting supernatant, measure viability by my Acanthaceous indigo (Biosource DALL 1100).

The result

Table 23: in the hBD2 existence and not, after LPS handles, from The peoplePeripheral blood lymphocytes (PBMC) produces TNF, and all specimen needle are tested identical donor, comes from two representativeness experiments in the donor.Measure TNF by cytometer beads array (CBA) on FACSarray, * * * refers to analyze (N=2) by double factor ANOVA, compares p＜0.001 with contrast separately.

Table 24: in the mBD3 existence and not, after LPS handles, from MicePeripheral blood lymphocytes (PBMC) produces TNF, and all specimen needle are tested identical donor, comes from two representativeness experiments in the donor.Measure TNF by cytometer beads array (CBA) on FACSarray, * * * refers to analyze (N=2) by double factor ANOVA, compares p＜0.001 with contrast separately.

As shown in Table 23, mice beta-defensin 3 (mBD3) is with the secretion of the degree downward modulation TNF identical with hBD2 and dexamethasone from the human PBMC.MBD3 also reduces the secretion (table 24) of TNF from mice PBMC.

Therefore, in this case, mBD3 represents splendid anti-inflammatory activity.

List of references

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Claims (15)

1. a mammal beta-defensin is used for the treatment of purposes in the medicine of rheumatoid arthritis in manufacturing.

2. according to the purposes of claim 1, wherein this mammal beta-defensin is that parenteral is used, and preferably is subcutaneous or intravenous is used.

3. according to each purposes among the claim 1-2, wherein this mammal beta-defensin system to about 100mg/kg body weight/day, the preferably about 0.1mg/kg body weight/day extremely dosage of about 10mg/kg body weight/day is used with about 0.1mg/kg body weight/day.

4. according to each purposes among the claim 1-3, wherein this mammal beta-defensin is a human beta-defensin.

5. according to each purposes among the claim 1-4, wherein the aminoacid sequence of this mammal beta-defensin and SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 has at least 80% homogeneity.

6. according to each purposes among the claim 1-5, wherein this human beta-defensin is human beta-defensin 1, human beta-defensin 2, human beta-defensin 3 or human beta-defensin 4.

7. according to each purposes among the claim 1-6, wherein the aminoacid sequence of this mammal beta-defensin and SEQID NO:2 has at least 80% homogeneity.

8. according to each purposes among the claim 1-7, wherein this mammal beta-defensin is a human beta-defensin 2.

9. method for the treatment of rheumatoid arthritis, this method comprises that the experimenter to the described treatment of needs uses the mammal beta-defensin of effective dose.

10. the method for claim 9, wherein this human beta-defensin is that parenteral is used, and preferably is subcutaneous or intravenous is used.

11. the method for claim 9, wherein this human beta-defensin or its functionally equivalent variant system to about 100mg/kg body weight, the preferably about 0.1mg/kg body weight extremely dosage of about 10mg/kg body weight is used with about 0.1mg/kg body weight.

12. the method for claim 9, wherein this mammal beta-defensin is a human beta-defensin.

13. the method for claim 9, wherein the aminoacid sequence of this mammal beta-defensin and SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 has at least 80% homogeneity.

14. the method for claim 9, wherein the aminoacid sequence of this mammal beta-defensin and SEQ ID NO:2 has at least 80% homogeneity.

15. the method for claim 9, wherein this human beta-defensin is human beta-defensin 1, human beta-defensin 2, human beta-defensin 3 or human beta-defensin 4.